This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Disorders of lysosome function lead to a variety of diseases including neurological dysfunction (lysosomal storage diseases) and osteopetrosis (overcalcification of bone). Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase, a counterion pathway. We recently used isolated lysosomes to identify and characterize a Chloride permeability in the lysosomal membrane which has the features required of such a counterion pathway and demonstrated that the chloride is transported by ClC-7, a Cl-/H+ antiporter specifically targeted to the lysosomal membrane. In the past year we have been developing methods to accurately meaure the pH in lysosomes in living cells in order to determine the influence of ClC-7 and other transporters on the lysosomal pH. These methods use dual-wavelength ratiometric fluorophores linked to dextran to specifically target lysosomes. pH is measured by processing images of the cells taken at the two wavelengths. This year, we have obtained preliminary confirmation that siRNA-based knockdown of ClC-7 inhibits lysosomal acidifciation and are refining the methods we are using to make these measurements. An alternate hypothesis regarding the role of ClC-7 is that it is important for determining the lumenal concentration of Cl- in lysosomes, which in turn is important for activating lysosomal degradative enzymes. We are testing this hypothesis using a variety of techniques. We have also begun to use fluorescence-based assays to measure the membrane voltage in isolated lysosomes. This is a useful to to analyze the relative contributions of different permeabilities to determining the lysosomal pH. To tie these approaches together, we, in collaboration with Dr. Michael Grabe at the University of Pittsburgh, are computationally simulating the known features of the lysosomal acidfication mecahnism to determine whether these can explain observed acdification behaviors. Together these methods provide an integrated approach to understanding the dynamics of lysosomal, and ultimatly organellar, pH regulation.